TREATMENT WITH ANTI ErbB2 ANTIBODIES

ABSTRACT

The present invention concerns the treatment of disorders characterized by the overexpression of ErbB2. More specifically, the invention concerns the treatment of human patients susceptible to or diagnosed with cancer overexpressing ErbB2 with a combination of an anti-ErbB2 antibody and a chemotherapeutic agent other than an anthracycline, e.g. doxorubicin or epirubicin. The invention further provides a method of treating cancer in a human patient comprising administering effective amounts of an anti-ErbB2 antibody and a cardioprotectant to the patient.

This is a continuation of non-provisional application Ser. No.09/209,023, filed Dec. 10, 1998 claiming priority to provisionalapplication No. 60/069,346, filed Dec. 12, 1997, the entire disclosuresof which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns the treatment of disorders characterizedby the overexpression of ErbB2. More specifically, the inventionconcerns the treatment of human patients susceptible to or diagnosedwith cancer overexpressing ErbB2 with a combination of an anti-ErbB2antibody and a chemotherapeutic agent other than an anthracycline, e.g.doxorubicin or epirubicin. The invention further provides a method oftreating cancer in a human patient comprising administering effectiveamounts of an anti-ErbB2 antibody and a cardioprotectant to the patient.

BACKGROUND OF THE INVENTION

Proto-oncogenes that encode growth factors and growth factor receptorshave been identified to play important roles in the pathogenesis ofvarious human malignancies, including breast cancer. It has been foundthat the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2),which encodes a 185-kd transmembrane glycoprotein receptor (p185^(HER2))related to the epidermal growth factor receptor (EGFR), is overexpressedin about 25% to 30% of human breast cancer (Slamon et al., Science235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]).

Several lines of evidence support a direct role for ErbB2 in thepathogenesis and clinical aggressiveness of ErbB2-overexpressing tumors.The introduction of ErbB2 into non-neoplastic cells has been shown tocause their malignant transformation (Hudziak et al., Proc. Natl. Acad.Sci. USA 84:7159-7163 [1987]; DiFiore et al., Science 237:178-182[1987]). Transgenic mice that express HER2 were found to develop mammarytumors (Guy et al., Proc. Natl. Acad. Sci.

Antibodies directed against human erbB2 protein products and proteinsencoded by the rat equivalent of the erbB2 gene (neu) have beendescribed. Drebin et al., Cell 41:695-706 (1985) refer to an IgG2amonoclonal antibody which is directed against the rat neu gene product.This antibody called 7.16.4 causes down-modulation of cell surface p185expression on B104-1-1 cells (NIH-3T3 cells transfected with the neuproto-oncogene) and inhibits colony formation of these cells. In Drebinet al. PNAS (USA) 83:9129-9133 (1986), the 7.16.4 antibody was shown toinhibit the tumorigenic growth of neu-transformed NIH-3T3 cells as wellas rat neuroblastoma cells (from which the neu oncogene was initiallyisolated) implanted into nude mice. Drebin et al. in Oncogene 2:387-394(1988) discuss the production of a panel of antibodies against the ratneu gene product. All of the antibodies were found to exert a cytostaticeffect on the growth of neu-transformed cells suspended in soft agar.Antibodies of the IgM, IgG2a and IgG2b isotypes were able to mediatesignificant in vitro lysis of neu-transformed cells in the presence ofcomplement, whereas none of the antibodies were able to mediate highlevels of antibody-dependent cellular cytotoxicity (ADCC) of theneu-transformed cells. Drebin et al. Oncogene 2:273-277 (1988) reportthat mixtures of antibodies reactive with two distinct regions on thep185 molecule result in synergistic anti-tumor effects onneu-transformed NIH-3T3 cells implanted into nude mice. Biologicaleffects of anti-neu antibodies are reviewed in Myers et al., Meth.Enzym. 198:277-290 (1991). See also WO94/22478 published Oct. 13, 1994.

Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe thegeneration of a panel of anti-ErbB2 antibodies which were characterizedusing the human breast tumor cell line SKBR3. Relative cellproliferation of the SKBR3 cells following exposure to the antibodieswas determined by crystal violet staining of the monolayers after 72hours. Using this assay, maximum inhibition was obtained with theantibody called 4D5 which inhibited cellular proliferation by 56%. Otherantibodies in the panel, including 7C2 and 7F3, reduced cellularproliferation to a lesser extent in this assay. Hudziak et al. concludethat the effect of the 4D5 antibody on SKBR3 cells was cytostatic ratherthan cytotoxic, since SKBR3 cells resumed growth at a nearly normal ratefollowing removal of the antibody from the medium. The antibody 4D5 wasfurther found to sensitize p185^(erbB2)-overexpressing breast tumor celllines to the cytotoxic effects of TNF-α. See also WO89/06692 publishedJul. 27, 1989. The anti-ErbB2 antibodies discussed in Hudziak et al. arefurther characterized in Fendly et al. Cancer Research 50:1550-1558(1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. GrowthRegulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol.11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986(1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993);Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. CancerResearch 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5(1991); and D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994).

Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe twoantibodies which were selected for their reactivity on the lungadenocarcinoma cell line (Calu-3) which overexpresses ErbB2. One of theantibodies, called MGR3, was found to internalize, inducephosphorylation of ErbB2, and inhibit tumor cell growth in vitro.

McKenzie et al. Oncogene 4:543-548 (1989) generated a panel ofanti-ErbB2 antibodies with varying epitope specificities, including theantibody designated TA1. This TA1 antibody was found to induceaccelerated endocytosis of ErbB2 (see Maier et al. Cancer Res.51:5361-5369 [1991]). Bacus et al. Molecular Carcinogenesis 3:350-362(1990) reported that the TA1 antibody induced maturation of the breastcancer cell lines AU-565 (which overexpresses the erbB2 gene) and MCF-7(which does not). Inhibition of growth and acquisition of a maturephenotype in these cells was found to be associated with reduced levelsof ErbB2 receptor at the cell surface and transient increased levels inthe cytoplasm.

Stancovski et al. PNAS (USA) 88:8691-8695 (1991) generated a panel ofanti-ErbB2 antibodies, injected them i.p. into nude mice and evaluatedtheir effect on tumor growth of murine fibroblasts transformed byoverexpression of the erbB2 gene. Various levels of tumor inhibitionwere detected for four of the antibodies, but one of the antibodies(N28) consistently stimulated tumor growth. Monoclonal antibody N28induced significant phosphorylation of the ErbB2 receptor, whereas theother four antibodies generally displayed low or nophosphorylation-inducing activity. The effect of the anti-ErbB2antibodies on proliferation of SKBR3 cells was also assessed. In thisSKBR3 cell proliferation assay, two of the antibodies (N12 and N29)caused a reduction in cell proliferation relative to control. Theability of the various antibodies to induce cell lysis in vitro viacomplement-dependent cytotoxicity (CDC) and antibody-mediatedcell-dependent cytotoxicity (ADCC) was assessed, with the authors ofthis paper concluding that the inhibitory function of the antibodies wasnot attributed significantly to CDC or ADCC.

Bacus et al. Cancer Research 52:2580-2589 (1992) further characterizedthe antibodies described in Bacus et al. (1990) and Stancovski et al. ofthe preceding paragraphs. Extending the i.p. studies of Stancovski etal., the effect of the antibodies after i.v. injection into nude miceharboring mouse fibroblasts overexpressing human ErbB2 was assessed. Asobserved in their earlier work, N28 accelerated tumor growth whereas N12and N29 significantly inhibited growth of the ErbB2-expressing cells.Partial tumor inhibition was also observed with the N24 antibody. Bacuset al. also tested the ability of the antibodies to promote a maturephenotype in the human breast cancer cell lines AU-565 and MDA-MB453(which overexpress ErbB2) as well as MCF-7 (containing low levels of thereceptor). Bacus et al. saw a correlation between tumor inhibition invivo and cellular differentiation; the tumor-stimulatory antibody N28had no effect on differentiation, and the tumor inhibitory action of theN12, N29 and N24 antibodies correlated with the extent ofdifferentiation they induced.

Xu et al. Int. J. Cancer 53:401-408 (1993) evaluated a panel ofanti-ErbB2 antibodies for their epitope binding specificities, as wellas their ability to inhibit anchorage-independent andanchorage-dependent growth of SKBR3 cells (by individual antibodies andin combinations), modulate cell-surface ErbB2, and inhibit ligandstimulated anchorage-independent growth. See also WO94/00136 publishedJan. 6, 1994 and Kasprzyk et al. Cancer Research 52:2771-2776 (1992)concerning anti-ErbB2 antibody combinations. Other anti-ErbB2 antibodiesare discussed in Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawveret al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res.54:3758-3765 (1994); and Harwerth et al. J. Biol. Chem. 267:15160-15167(1992).

A recombinant humanized anti-ErbB2 monoclonal antibody (a humanizedversion of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAbHER2 or HERCEPTIN®) has been clinically active in patients withErbB2-overexpressing metastatic breast cancers that had receivedextensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol.14:737-744 [1996]).

ErbB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene 159:19-27 [1995]; and Hynes and Stern, BiochimBiophys Acta 1198:165-184 [1994]), and has been linked to sensitivityand/or resistance to hormone therapy and chemotherapeutic regimens,including CMF (cyclophosphamide, methotrexate, and fluoruracil) andanthracyclines (Baselga et al., Oncology 11(3 Suppl 2):43-48 [1997]).However, despite the association of ErbB2 overexpression with poorprognosis, the odds of HER2-positive patients responding clinically totreatment with taxanes were greater than three times those ofHER2-negative patients (Ibid). rhuMab HER2 was shown to enhance theactivity of paclitaxel (TAXOL®) and doxorubicin against breast cancerxenografts in nude mice injected with BT-474 human breast adenocarcinomacells, which express high levels of HER2 (Baselga et al., Breast Cancer,Proceedings of ASCO, Vol. 13, Abstract 53 [1994]).

SUMMARY OF THE INVENTION

The present invention concerns the treatment of disorders characterizedby overexpression of ErbB2, and is based on the recognition that whiletreatment with anti-ErbB2 antibodies markedly enhances the clinicalbenefit of the use of chemotherapeutic agents in general, a syndrome ofmyocardial dysfunction that has been observed as a side-effect ofanthracycline derivatives is increased by the administration ofanti-ErbB2 antibodies.

Accordingly, the invention concerns a method for the treatment of ahuman patient susceptible to or diagnosed with a disorder characterizedby overexpression of ErbB2 receptor comprising administering atherapeutically effective amount of a combination of an anti-ErbB2antibody and a chemotherapeutic agent other than an anthracyclinederivative, e.g. doxorubicin or epirubicin, in the absence of ananthracycline derivative, to the human patient.

The disorder preferably is a benign or malignant tumor characterized bythe overexpression of the ErbB2 receptor, e.g. a cancer, such as, breastcancer, squamous cell cancer, small-cell lung cancer, non-small celllung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, bladder cancer, hepatoma, colon cancer,colorectal cancer, endometrial carcinoma, salivary gland carcinoma,kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer. Thechemotherapeutic agent preferably is a taxoid, such as TAXOL®(paclitaxel) or a TAXOL® derivative.

Although an antiproliferative effect is sufficient, in a preferredembodiment, the anti-ErbB2 antibody is capable of inducing cell death oris capable of inducing apoptosis. Preferred anti-ErbB2 antibodies bindthe extracellular domain of the ErbB2 receptor, and preferably bind tothe epitope 4D5 or 3H4 within the ErbB2 extracellular domain sequence.More preferably, the antibody is the antibody 4D5, most preferably in ahumanized form.

The method of the present invention is particularly suitable for thetreatment of breast or ovarian cancer, characterized by theoverexpression of the ErbB2 receptor.

In another aspect, the invention concerns an article of manufacture,comprising a container, a composition within the container comprising ananti-ErbB2 antibody, optionally a label on or associated with thecontainer that indicates that the composition can be used for treating acondition characterized by overexpression of ErbB2 receptor, and apackage insert containing instructions to avoid the use ofanthracycline-type chemotherapeutics in combination with thecomposition.

In a further embodiment, the invention provides a method of treatingErbB2 expressing cancer in a human patient comprising administeringeffective amounts of an anti-ErbB2 antibody and a cardioprotectant tothe patient. The cancer is preferably characterized by overexpression ofErbB2. In one embodiment, the method further comprises administering ananthracyline antibiotic to the patient. In another embodiment, ananthracycline antibiotic is not administered to the patient with theanti-ErbB2 antibody or cardioprotectant. One or more additionalchemotherapeutic agents may also be administered to the patient.

The invention further provides an article of manufacture comprising acontainer, a composition within the container comprising an anti-ErbB2antibody and a package insert instructing the user of the composition toadminister the anti-ErbB2 antibody composition and a cardioprotectant toa patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows epitope-mapping of the extracellular domain of ErbB2 asdetermined by truncation mutant analysis and site-directed mutagenesis(Nakamura et al. J. of Virology 67(10):6179-6191 [October 1993]; Renz etal. J. Cell Biol. 125(6):1395-1406 [June 1994]). The anti-proliferativeMAbs 4D5 and 3H4 bind adjacent to the transmembrane domain. The variousErbB2-ECD truncations or point mutations were prepared from cDNA usingpolymerase chain reaction technology. The ErbB2 mutants were expressedas gD fusion proteins in a mammalian expression plasmid. This expressionplasmid uses the cytomegalovirus promoter/enhancer with SV40 terminationand polyadenylation signals located downstream of the inserted cDNA.Plasmid DNA was transfected into 293S cells. One day followingtransfection, the cells were metabolically labeled overnight inmethionine and cysteine-free, low glucose DMEM containing 1% dialyzedfetal bovine serum and 25 μCi each of ³⁵S methionine and ³⁵S cysteine.Supernatants were harvested either the ErbB2 MAbs or control antibodieswere added to the supernatant and incubated 2-4 hours at 4° C. Thecomplexes were precipitated, applied to a 10-20% Tricine SDS gradientgel and electrophoresed at 100 V. The gel was electroblotted onto amembrane and analyzed by autoradiography. SEQ ID NOs:8 and 9 depict the3H4 and 4D5 epitopes, respectively.

FIG. 2 depicts with underlining the amino acid sequence of Domain 1 ofErbB2 (SEQ ID NO:1). Bold amino acids indicate the location of theepitope recognized by MAbs 7C2 and 7F3 as determined by deletionmapping, i.e. the “7C2/7F3 epitope” (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “HER2”, “ErbB2” “c-Erb-B2” are used interchangeably. Unlessindicated otherwise, the terms “ErbB2”, “c-Erb-B2” and “HER2” when usedherein refer to the human protein and “her2”, “erbB2” and “c-erb-B2”refer to human gene. The human erbB2 gene and ErbB2 protein are, forexample, described in Semba et al., PNAS (USA) 82:6497-6501 (1985) andYamamoto et al. Nature 319:230-234 (1986) (Genebank accession numberX03363). ErbB2 comprises four domains (Domains 1-4).

The “epitope 4D5” is the region in the extracellular domain of ErbB2 towhich the antibody 4D5 (ATCC CRL 10463) binds. This epitope is close tothe transmembrane region of ErbB2. To screen for antibodies which bindto the 4D5 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed (see FIG. 1) to assesswhether the antibody binds to the 4D5 epitope of ErbB2 (i.e. any one ormore residues in the region from about residue 529, e.g. about residue561 to about residue 625, inclusive).

The “epitope 3H4” is the region in the extracellular domain of ErbB2 towhich the antibody 3H4 binds. This epitope is shown in FIG. 1, andincludes residues from about 541 to about 599, inclusive, in the aminoacid sequence of ErbB2 extracellular domain.

The “epitope 7C2/7F3” is the region at the N terminus of theextracellular domain of ErbB2 to which the 7C2 and/or 7F3 antibodies(each deposited with the ATCC, see below) bind. To screen for antibodieswhich bind to the 7C2/7F3 epitope, a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to establish whether theantibody binds to the 7C2/7F3 epitope on ErbB2 (i.e. any one or more ofresidues in the region from about residue 22 to about residue 53 ofErbB2; SEQ ID NO:2).

The term “induces cell death” or “capable of inducing cell death” refersto the ability of the antibody to make a viable cell become nonviable.The “cell” here is one which expresses the ErbB2 receptor, especiallywhere the cell overexpresses the ErbB2 receptor. A cell which“overexpresses” ErbB2 has significantly higher than normal ErbB2 levelscompared to a noncancerous cell of the same tissue type. Preferably, thecell is a cancer cell, e.g. a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. In vitro, the cell may be a SKBR3, BT474, Calu 3, MDA-MB-453,MDA-MB-361 or SKOV3 cell. Cell death in vitro may be determined in theabsence of complement and immune effector cells to distinguish celldeath induced by antibody dependent cellular cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC). Thus, the assay for cell deathmay be performed using heat inactivated serum (i.e. in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody is able to induce cell death, loss of membraneintegrity as evaluated by uptake of propidium iodide (PI), trypan blue(see Moore et al. Cytotechnology 17:1-11 [1995]) or 7AAD can be assessedrelative to untreated cells. Preferred cell death-inducing antibodiesare those which induce PI uptake in the “PI uptake assay in BT474cells”.

The phrase “induces apoptosis” or “capable of inducing apoptosis” refersto the ability of the antibody to induce programmed cell death asdetermined by binding of annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell isone which overexpresses the ErbB2 receptor. Preferably the “cell” is atumor cell, e.g. a breast, ovarian, stomach, endometrial, salivarygland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Invitro, the cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453,MDA-MB-361 or SKOV3 cell. Various methods are available for evaluatingthe cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNAfragmentation can be evaluated through DNA laddering as disclosed in theexample herein; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcell in an “annexin binding assay using BT474 cells” (see below).

Sometimes the pro-apoptotic antibody will be one which blocks HRGbinding/activation of the ErbB2/ErbB3 complex (e.g. 7F3 antibody). Inother situations, the antibody is one which does not significantly blockactivation of the ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2).Further, the antibody may be one like 7C2 which, while inducingapoptosis, does not induce a large reduction in the percent of cells inS phase (e.g. one which only induces about 0-10% reduction in thepercent of these cells relative to control).

The antibody of interest may be one like 7C2 which binds specifically tohuman ErbB2 and does not significantly cross-react with other proteinssuch as those encoded by the erbB1, erbB3 and/or erbB4 genes. Sometimes,the antibody may not significantly cross-react with the rat neu protein,e.g., as described in Schecter et al. Nature 312:513 (1984) and Drebinet al., Nature 312:545-548 (1984). In such embodiments, the extent ofbinding of the antibody to these proteins (e.g., cell surface binding toendogenous receptor) will be less than about 10% as determined byfluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA).

“Heregulin” (HRG) when used herein refers to a polypeptide whichactivates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes (i.e.induces phosphorylation of tyrosine residues in the complex upon bindingthereto). Various heregulin polypeptides encompassed by this term aredisclosed in Holmes et al., Science, 256:1205-1210 (1992); WO 92/20798;Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994); and Marchionni etal., Nature, 362:312-318 (1993), for example. The term includesbiologically active fragments and/or variants of a naturally occurringHRG polypeptide, such as an EGF-like domain fragment thereof (e.g.HRGβ1₁₇₇₋₂₄₄).

The “ErbB2-ErbB3 protein complex” and “ErbB2-ErbB4 protein complex” arenoncovalently associated oligomers of the ErbB2 receptor and the ErbB3receptor or ErbB4 receptor, respectively. The complexes form when a cellexpressing both of these receptors is exposed to HRG and can be isolatedby immunoprecipitation and analyzed by SDS-PAGE as described inSliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994).

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework region (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRsand, with the CDRs from the other chain, contribute to the formation ofthe antigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91-3242, Vol. I, pages 647-669 [1991]). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng.8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementaritydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit having the desired specificity, affinity, and capacity. In someinstances, Fv framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues which are found neither in therecipient antibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and maximize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522-525 (1986); Reichmannet al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992). The humanized antibody includes a PRIMATIZED™ antibodywherein the antigen-binding region of the antibody is derived from anantibody produced by immunizing macaque monkeys with the antigen ofinterest.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

A “disorder” is any condition that would benefit from treatment with theanti-ErbB2 antibody. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include benign and malignant tumors; leukemias andlymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

The term “therapeutically effective amount” is used to refer to anamount having antiproliferative effect. Preferably, the therapeuticallyeffective amount has apoptotic activity, or is capable of inducing celldeath, and preferably death of benign or malignant tumor cells, inparticular cancer cells. Efficacy can be measured in conventional ways,depending on the condition to be treated. For cancer therapy, efficacycan, for example, be measured by assessing the time to diseaseprogression (TTP), or determining the response rates (RR) (see theExample below).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma and various types of head and neck cancer.

An “ErbB2-expressing cancer” is one comprising cells which have ErbB2protein present at their cell surface, such that an anti-ErbB2 antibodyis able to bind to the cancer.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carzinophilin,chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin, epirubicin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folicacid analogues such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine, 5-FU; androgens such ascalusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elformithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;caminomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid;esperamicins; capecitabine; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. Also included in this definition arehormonal agents that act to regulate or inhibit hormone action on tumorssuch as anti-estrogens including for example tamoxifen, raloxifene,aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone; and anti-androgens such as flutamideand nilutamide; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially anErbB2-overexpressing cancer cell either in vitro or in vivo. Thus, thegrowth inhibitory agent is one which significantly reduces thepercentage of ErbB2 overexpressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), TAXOL®, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The 4D5 antibody (and functionalequivalents thereof) can also be employed for this purpose.

“Doxorubicin” is an athracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.Other anthracycline antibiotics include epirubicin, daunorubicin,caminomycin, detorubicin, esorubicin, marcellomycin, quelamycin,rodorubicin, idarubicin, as well as pharmaceutically active salts, acidsor derivatives of any of these.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

By “solid phase” is meant a non-aqueous matrix to which the antibodiesused in accordance with the present invention can adhere. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-ErbB2 antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

A “cardioprotectant” is a compound or composition which prevents orreduces myocardial dysfunction (i.e. cardiomyopathy and/or congestiveheart failure) associated with administration of a drug, such as ananthracycline antibiotic and/or an anti-ErbB2 antibody, to a patient.The cardioprotectant may, for example, block or reduce afree-radical-mediated cardiotoxic effect and/or prevent or reduceoxidative-stress injury. Examples of cardioprotectants encompassed bythe present definition include the iron-chelating agent dexrazoxane(ICRF-187) (Seifert et al. The Annals of Pharmacotherapy 28:1063-1072[1994]); a lipid-lowering agent and/or anti-oxidant such as probucol(Singal et al. J. Mol. Cell Cardiol. 27:1055-1063 [1995]); amifostine(aminothiol 2-[(3-aminopropyl)amino]ethanethiol-dihydrogen phosphateester, also called WR-2721, and the dephosphorylated cellular uptakeform thereof called WR-1065) andS-3-(3-methylaminopropylamino)propylphosphorothioic acid (WR-151327),see Green et al. Cancer Research 54:738-741 (1994); digoxin (Bristow, M.R. In: Bristow M R, ed. Drug-Induced Heart Disease. New York: Elsevier191-215 [1980]); beta-blockers such as metoprolol (Hjalmarson et al.Drugs 47:Suppl 4:31-9 [1994]; and Shaddy et al. Am. Heart J. 129:197-9[1995]); vitamin E; ascorbic acid (vitamin C); free radical scavengerssuch as oleanolic acid, ursolic acid and N-acetylcysteine (NAC); spintrapping compounds such as alpha-phenyl-tert-butyl nitrone (PBN);(Paracchini et al., Anticancer Res. 13:1607-1612 [1993]); selenoorganiccompounds such as P251 (Elbesen); and the like.

II. Production of Anti-ErbB2 Antibodies

A description follows as to exemplary techniques for the production ofthe antibodies used in accordance with the present invention. The ErbB2antigen to be used for production of antibodies may be, e.g., a solubleform of the extracellular domain of ErbB2 or a portion thereof,containing the desired epitope. Alternatively, cells expressing ErbB2 attheir cell surface (e.g. NIH-3T3 cells transformed to overexpress ErbB2;or a carcinoma cell line such as SKBR3 cells, see Stancovski et al. PNAS(USA) 88:8691-8695 [1991]) can be used to generate antibodies. Otherforms of ErbB2 useful for generating antibodies will be apparent tothose skilled in the art.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with 1/5 to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63[Marcel Dekker, Inc., New York, 1987]).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103[Academic Press, 1986]). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 [1992]), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 [1993]). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 [1984]), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized and Human Antibodies

Methods for humanizing non-human antibodies are well known in the art.Preferably, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 [1988]), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901[1987]). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 [1993]).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 [1991]).

(iv) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 [1985]). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 [1992]). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

(v) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the ErbB2 protein. For example, onearm may bind an epitope in Domain 1 of ErbB2 such as the 7C2/7F3epitope, the other may bind a different ErbB2 epitope, e.g. the 4D5epitope. Other such antibodies may combine an ErbB2 binding site withbinding site(s) for EGFR, ErbB3 and/or ErbB4. Alternatively, ananti-ErbB2 arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 orCD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms tothe ErbB2-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express ErbB2. These antibodiespossess an ErbB2-binding arm and an arm which binds the cytotoxic agent(e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 [1983]). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vi) Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. Thoseantibodies having the characteristics described herein are selected.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., PI, trypan blue or 7AAD uptake isassessed relative to control. The preferred assay is the “PI uptakeassay using BT474 cells”. According to this assay, BT474 cells (whichcan be obtained from the American Type Culture Collection [Rockville,Md.]) are cultured in Dulbecco's Modified Eagle Medium (D-MEM):Ham'sF-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine. (Thus, the assay is performed in the absence ofcomplement and immune effector cells). The BT474 cells are seeded at adensity of 3×10⁶ per dish in 100×20 mm dishes and allowed to attachovernight. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the appropriate MAb. The cellsare incubated for a 3 day time period. Following each treatment,monolayers are washed with PBS and detached by trypsinization. Cells arethen centrifuged at 1200 rpm for 5 minutes at 4° C., the pelletresuspended in 3 ml ice cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4,140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake areselected.

In order to select for antibodies which induce apoptosis, an “annexinbinding assay using BT474 cells” is available. The BT474 cells arecultured and seeded in dishes as discussed in the preceding paragraph.The medium is then removed and replaced with fresh medium alone ormedium containing 10 μg/ml of the MAb. Following a three day incubationperiod, monolayers are washed with PBS and detached by trypsinization.Cells are then centrifuged, resuspended in Ca²⁺ binding buffer andaliquoted into tubes as discussed above for the cell death assay. Tubesthen receive labeled annexin (e.g. annexin V-FTIC) (1 μg/ml). Samplesmay be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). Those antibodies which inducestatistically significant levels of annexin binding relative to controlare selected as apoptosis-inducing antibodies.

In addition to the annexin binding assay, a “DNA staining assay usingBT474 cells” is available. In order to perform this assay, BT474 cellswhich have been treated with the antibody of interest as described inthe preceding two paragraphs are incubated with 9 μg/ml HOECHST 33342™for 2 hr at 37° C., then analyzed on an EPICS ELITE™ flow cytometer(Coulter Corporation) using MODFIT LT™ software (Verity Software House).Antibodies which induce a change in the percentage of apoptotic cellswhich is 2 fold or greater (and preferably 3 fold or greater) thanuntreated cells (up to 100% apoptotic cells) may be selected aspro-apoptotic antibodies using this assay.

To screen for antibodies which bind to an epitope on ErbB2 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed by methods known in theart.

To identify anti-ErbB2 antibodies which inhibit growth of SKBR3 cells incell culture by 50-100%, the SKBR3 assay described in WO89/06692 can beperformed. According to this assay, SKBR3 cells are grown in a 1:1mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum,glutamine and penicillinstreptomycin. The SKBR3 cells are plated at20,000 cells in a 35 mm cell culture dish (2 mls/35 mm dish). 2.5 μg/mlof the anti-ErbB2 antibody is added per dish. After six days, the numberof cells, compared to untreated cells are counted using an electronicCOULTER™ cell counter. Those antibodies which inhibit growth of theSKBR3 cells by 50-100% are selected for combination with the apoptoticantibodies as desired.

(vii) Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of the antibodyin treating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

(viii) Immunoconjugates

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-ErbB2 antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

(ix) Immunoliposomes

The anti-ErbB2 antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

(x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 [1987]). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-ErbB2antibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 [1984]).

(xi) Antibody-Salvage Receptor Binding Epitope Fusions

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half life. This maybe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment (e.g. by mutation of the appropriateregion in the antibody fragment or by incorporating the epitope into apeptide tag that is then fused to the antibody fragment at either end orin the middle, e.g., by DNA or peptide synthesis).

A systematic method for preparing such an antibody variant having anincreased in vivo half-life comprises several steps. The first involvesidentifying the sequence and conformation of a salvage receptor bindingepitope of an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the antibody of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the antibody variant is tested to see ifit has a longer in vivo half-life than that of the original antibody. Ifthe antibody variant does not have a longer in vivo half-life upontesting, its sequence is further altered to include the sequence andconformation of the identified binding epitope. The altered antibody istested for longer in vivo half-life, and this process is continued untila molecule is obtained that exhibits a longer in vivo half-life.

The salvage receptor binding epitope being thus incorporated into theantibody of interest is any suitable such epitope as defined above, andits nature will depend, e.g., on the type of antibody being modified.The transfer is made such that the antibody of interest still possessesthe biological activities described herein.

The epitope preferably constitutes a region wherein any one or moreamino acid residues from one or two loops of a Fc domain are transferredto an analogous position of the antibody fragment. Even more preferably,three or more residues from one or two loops of the Fc domain aretransferred. Still more preferred, the epitope is taken from the CH2domain of the Fc region (e.g., of an IgG) and transferred to the CH1,CH3, or V_(H) region, or more than one such region, of the antibody.Alternatively, the epitope is taken from the CH2 domain of the Fc regionand transferred to the C_(L) region or V_(L) region, or both, of theantibody fragment.

In one most preferred embodiment, the salvage receptor binding epitopecomprises the sequence (5′ to 3′): PKNSSMISNTP (SEQ ID NO:3), andoptionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO:4), HQNLSDGK (SEQ ID NO:5), HQNISDGK(SEQ ID NO:6), or VISSHLGQ (SEQ ID NO:7), particularly where theantibody fragment is a Fab or F(ab′)₂. In another most preferredembodiment, the salvage receptor binding epitope is a polypeptidecontaining the sequence(s) (5′ to 3′): HQNLSDGK (SEQ ID NO:5), HQNISDGK(SEQ ID NO:6), or VISSHLGQ (SEQ ID NO:7) and the sequence: PKNSSMISNTP(SEQ ID NO:3).

(xii) Purification of Anti-ErbB2 Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems arepreferably first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 [1983]). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 [1986]). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g. from about 0-0.25M salt).

III. Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized anti-ErbB2 antibodyformulations are described in WO 97/04801, expressly incorporated hereinbe reference.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to EGFR, ErbB2 (e.g. an antibody which binds a different epitope onErbB2), ErbB3, ErbB4, or vascular endothelial factor (VEGF) in the oneformulation. Alternatively, or in addition, the composition may comprisea cytotoxic agent, cytokine, growth inhibitory agent and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

IV. Treatment with the Anti-ErbB2 Antibodies

It is contemplated that, according to the present invention, theanti-ErbB2 antibodies may be used to treat various conditionscharacterized by overexpression and/or activation of the ErbB2 receptor.Exemplary conditions or disorders include benign or malignant tumors(e.g. renal, liver, kidney, bladder, breast, gastric, ovarian,colorectal, prostate, pancreatic, lung, vulval, thyroid, hepaticcarcinomas; sarcomas; glioblastomas; and various head and neck tumors);leukemias and lymphoid malignancies; other disorders such as neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologicdisorders.

The antibodies, chemotherapeutic agents and cardioprotectants of theinvention are administered to a human patient, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. Intravenous orsubcutaneous administration of the antibody is preferred.

In one embodiment, the treatment of the present invention involves thecombined administration of an anti-ErbB2 antibody and a chemotherapeuticagent, other than an anthracycline derivative. The present inventioncontemplates administration of cocktails of different chemotherapeuticagents. The combined administration includes coadministration, usingseparate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992). The chemotherapeutic agent may precede, or followadministration of the antibody or may be given simultaneously therewith.The antibody may be combined with an anti-estrogen compound such astamoxifen or an anti-progesterone such as onapristone (see, EP 616 812)in dosages known for such molecules.

It may be desirable to also administer antibodies against other tumorassociated antigens, such as antibodies which bind to the EGFR, ErbB3,ErbB4, or vascular endothelial factor (VEGF). Alternatively, or inaddition, two or more anti-ErbB2 antibodies may be co-administered tothe patient. Sometimes, it may be beneficial to also administer one ormore cytokines to the patient. In a preferred embodiment, the ErbB2antibody is co-administered with a growth inhibitory agent. For example,the growth inhibitory agent may be administered first, followed by theErbB2 antibody. However, simultaneous administration or administrationof the ErbB2 antibody first is also contemplated. Suitable dosages forthe growth inhibitory agent are those presently used and may be lowereddue to the combined action (synergy) of the growth inhibitory agent andanti-ErbB2 antibody.

The present invention also provides a method of treating ErbB2expressing cancer, especially cancer characterized by overexpression ofErbB2, by administering effective amounts of an anti-ErbB2 antibody anda cardioprotectant to a patient susceptible to, or diagnosed with,cancer.

In one embodiment, the method further comprises administering one ormore chemotherapeutic agents to the patient. For example, ananthracycline antibiotic may be administered to the patient along withthe cardioprotectant and anti-ErbB2 antibody (as well as one or moreother optional chemotherapeutic agents).

In an alternative embodiment, an anthracycline antibiotic is notadministered to the patient with the anti-ErbB2 antibody orcardioprotectant. Thus, while the patient may have been exposed to ananthracycline antibiotic in a previous treatment regimen, in thisembodiment of the invention, the therapeutic regimen does not entail theadministration of an antracycline antibiotic (i.e. the method isperformed in the absence of an anthracycline antibiotic). Thisembodiment of the invention contemplates however the optionaladministration of one or more other chemotherapeutic agents (other thananthracycline antibiotics) to the patient.

Combined administration of the anti-ErbB2 antibody and cardioprotectantincludes coadministration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.Moreover, administration of the cardioprotectant may be delayed untilthe patient has been given a lifetime dose of anti-ErbB2 antibody and/oranthracycline antibiotic which is considered to result in a high risk ofcardiac side effects (e.g. a lifetime doxorubicin dose of 350 mg/m²). Inone embodiment, the cardioprotectant is administered to the patient bothprior to administration of the anti-ErbB2 antibody and concurrentlytherewith.

The effective amount of the cardioprotectant is that which reduces orprevents myocardial dysfunction (e.g. cardiomyopathy and/or congestiveheart failure) resulting from administration of the anti-ErbB2 antibody(and optionally an anthracycline antibiotic or other chemotherapeuticagent) to the patient. Myocardial dysfunction can be assessed by avariety of different methods including physical examination,electrocardiography, echocardiography, angiocardiography, andendomyocardial biopsy (reviewed in Singal et al. NEJM 339:900-905(1998), expressly incorporated herein by reference). The preferredmethod for determining myocardial dysfunction comprises measuring theejection fraction by echocardiography or angiocardiography. Suitabledosages for the cardioprotectants dexrazoxane and probucol have beendescribed, for example, in Seifert et al. The Annals of Pharmacotherapy28:1063-1072 (1994) and Singal et al. J. Mol. Cell Cardiol. 27:1055-1063(1995), respectively. The effective amount of the cardioprotectant maybe determined based on the amount of anti-ErbB2 antibody and/oranthracycline antibiotic agent administered to the patient. Generally,the amount of cardioprotectant will exceed the amount ofchemotherapeutic agent or anti-ErbB2 antibody causing thecardiotoxicity. For example, the ratio of cardioprotectant:antibody orchemotherapeutic agent may be from about 1:1 to about 100:1.

In addition to the above therapeutic regimes, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

Further information about suitable dosages is provided in the Examplebelow.

V. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container, alabel and a package insert. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is ananti-ErbB2 antibody. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes. In addition, the article of manufacture comprises a packageinserts with instructions for use, including for example a warning thatthe composition is not to be used in combination with anthacycline-typechemotherapeutic agent, e.g. doxorubicin, or epirubicin, or instructingthe user of the composition to administer the anti-ErbB2 antibodycomposition and a cardioprotectant to a patient.

Deposit of Materials

The following hybridoma cell lines have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC):

Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 Oct. 17,1996 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRL 10463 May 24, 1990

Further details of the invention are illustrated by the followingnon-limiting Example.

Example Materials and Methods

Anti-ErbB2 Monoclonal Antibody

The anti-ErbB2 IgG₁κ murine monoclonal antibody 4D5, specific for theextracellular domain of ErbB2, was produced as described in Fendly etal., Cancer Research 5o:1550-1558 (1990) and WO89/06692. Briefly, NIH3T3/HER2-3₄₀₀ cells (expressing approximately 1×10⁵ ErbB2molecules/cell) produced as described in Hudziak et al. Proc. Natl.Acad. Sci. (USA) 84:7159 (1987) were harvested with phosphate bufferedsaline (PBS) containing 25 mM EDTA and used to immunize BALB/c mice. Themice were given injections i.p. of 10⁷ cells in 0.5 ml PBS on weeks, 0,2, 5 and 7. The mice with antisera that immunoprecipitated ³²P-labeledErbB2 were given i.p. injections of a wheat germ agglutinin-Sepharose(WGA) purified ErbB2 membrane extract on weeks 9 and 13. This wasfollowed by an i.v. injection of 0.1 ml of the ErbB2 preparation and thesplenocytes were fused with mouse myeloma line X63-Ag8.653. Hybridomasupernatants were screened for ErbB2-binding by ELISA andradioimmunoprecipitation. MOPC-21 (IgG1), (Cappell, Durham, N.C.), wasused as an isotype-matched control.

The treatment was performed with a humanized version of the murine 4D5antibody (HERCEPTIN®). The humanized antibody was engineered byinserting the complementarity determining regions of the murine 4D5antibody into the framework of a consensus human immunoglobulin IgG₁(IgG₁) (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 [1992]).The resulting humanized anti-ErbB2 monoclonal antibody has high affinityfor p185^(HER2) (Dillohiation constant [K_(d)]=0.1 nmol/L), markedlyinhibits, in vitro and in human xenografts, the growth of breast cancercells that contain high levels of p185^(HER2), inducesantibody-dependent cellular cytotoxicity (ADCC), and has been foundclinically active, as a single agent, in patients withErbB2-overexpressing metastatic breast cancers that had receivedextensive prior therapy. HERCEPTIN® is produced by a geneticallyengineered Chinese Hamster Ovary (CHO) cell line, grown in large scale,that secretes the antibody into the culture medium. The antibody ispurified from the CHO culture media using standard chromatographic andfiltration methods. Each lot of antibody used in this study was assayedto verify identity, purity, and potency, as well as to meet Food andDrug Administration requirements for sterility and safety.

Eligibility Criteria

Patients had to fulfill all of the following criteria to be eligible forstudy admission:

-   -   Metastatic breast cancer    -   Overexpression of the ErbB2 (HER2) oncogene (2+ to 3+ as        determined by immunohistochemistry or fluorescence in situ        hybridization (FISH). [Tumor expression of ErbB2 can be        determined by immunohistochemical analysis, as previously        described (Slamon et al., [1987] and [1989], supra), of a set of        thin sections prepared from the patient's paraffin-archived        tumor blocks. The primary detecting antibody used is murine 4D5        MAb, which has the same CDRs as the humanized antibody used for        the treatment. Tumors are considered to overexpress ErbB2 if at        least 25% of tumor cells exhibit characteristic membrane        staining for p185^(HER2)].    -   Bidimensionally measurable disease (including lytic bone        lesions) by radiographic means, physical examination, or        photographs

Measurable disease was defined as any mass reproducibly measurable intwo perpendicular diameters by physical examination, X-ray (plainfilms), computerized tomography (CT), magnetic resonance imaging (MRI),ultrasound, or photographs.

Osteoblastic metastases, pleural effusions, or ascites were notconsidered to be measurable. Measurable lesions must be at least 1 cm ingreatest dimension. Enumeration of evaluable sites of metastatic diseaseand number of lesions in an evaluable site (e.g. lung) had to berecorded on the appropriate Case Report Form (CRF). If a large number ofpulmonary or hepatic lesions were present, the six largest lesions persite were followed.

-   -   The ability to understand and willingness to sign a written        informed consent form    -   Women ≧18 years    -   Suitable candidates for receiving concomitant cytotoxic        chemotherapy as evidenced by screening laboratory assessments of        hematologic, renal, hepatic, and metabolic functions.

Exclusion Criteria

Patients with any of the following were excluded from study entry:

-   -   Prior cytotoxic chemotherapy for metastatic breast cancer        Patients may have received prior hormonal therapy (e.g.        tamoxifen) for metastatic disease or cytotoxic therapy in the        adjuvant setting.    -   Concomitant malignancy that has not been curatively treated    -   A performance status of <60% on the Karnofsky scale    -   Pregnant or nursing women; women of childbearing potential,        unless using effective contraception as determined by the        investigator    -   Bilateral breast cancer (either both primary tumors must have 2+        to 3+ HER2 overexpression, or the metastatic site must have 2+        to 3+ HER2 overexpression)    -   Use of investigational or unlicensed agents within 30 days prior        to study entry    -   Clinically unstable or untreated metastases to the brain (e.g.        requiring radiation therapy)

Based upon the foregoing criteria, 469 patients were chosen, andenrolled in the study. Half the patients (stratified by chemotherapy)were randomized to additionally receive the HERCEPTIN® antibody (seebelow).

Administration and Dosage

Anti-ErbB2 Antibody

On day 0, a 4 mg/kg dose of humanized anti-ErbB2 antibody (HERCEPTIN®,H) was administered intravenously; over a 90-minute period. Beginning onday 7, patients received weekly administration of 2 mg/kg antibody(i.v.) over a 90-minute period.

Chemotherapy

The patients received one of two chemotherapy regiments for a minimum ofsix cycles, provided their disease was not progressing: a)cyclophosphamide and doxorubicin or epirubicin (AC), if patients havenot received anthracycline therapy in the adjuvant setting, or b)paclitaxel (T, TAXOL®), if patients have received any anthracyclinetherapy in the adjuvant setting. The initial dose of the HERCEPTIN®antibody preceded the first cycle of either chemotherapy regimen by 24hours. Subsequent doses of the antibody were given immediately beforechemotherapy administration, if the initial dose of the antibody waswell tolerated. If the first dose of the antibody was not welltolerated, subsequent infusions continued to precede chemotherapyadministration by 24 hours. Patients were permitted to continuereceiving chemotherapy beyond six cycles if, in the opinion of thetreating physician, they were continuing to receive treatment benefit.

Cyclophosphamide (600 mg/m²) was given either by iv push over a minimumperiod of 3 minutes or by infusion over a maximum period of 2 hours.

Doxorubicin (60 mg/m²) or epirubicin (75 mg/m²) were given either byslow iv push over a minimum period of 3-5 minutes or by infusion over amaximum period of 2 hours, according to institutional protocol.

Paciltaxel (TAXOL®) was given at a dose of 175 mg/m² over 3 hours byintravenous administration. All patients receiving paclitaxel werepremedicated with dexamethasone (or its equivalent) 20 mg×2,administered orally 12 and 6 hours prior to paclitaxel; diphenhydramine(or its equivalent) 50 mg, iv, administered 30 minutes prior topaclitaxel, and dimetidine (or another H₂ blocker) 300 mg, iv,administered 30 minutes prior to paclitaxel.

Response Criteria

Progressive Disease

Objective evidence of an increase of 25% or more in any measurablelesion. Progressive disease also includes those instances when newlesions have appeared. For bone lesions, progression is defined as a 25%increase in objective measurement by plain film, CT, MRI; symptomaticnew lesions not due to fracture; or requirement for palliativeradiotherapy.

Complete Response

Disappearance of all radiographically and/or visually apparent tumor fora minimum of 4 weeks. Skin and chest wall complete responses had to beconfirmed by biopsy.

Partial Response

A reduction of at least 50% in the sum of the products of theperpendicular diameters of all measurable lesions for a minimum periodof 4 weeks. No new lesions may have appeared, nor may any lesions haveprogressed in size.

Minor Response

A reduction of 25% to 49% in the sum of the products of theperpendicular diameters of all measurable lesions. No new lesions mayhave appeared, nor may any lesions have progressed in size.

Stable Disease

No change of greater than 25% in the size of measurable lesions. Nolesions may have appeared.

Time to disease progression (TTP) was calculated from the beginning oftherapy to progression. Confidence limits for response rates werecalculated using the exact method for a single proportion. (Fleiss, J L,Statistical Methods for Rates and Proportions (ed.2), New York, N.Y.,Wiley, 1981, pp 13-17).

Results

At a median follow-up of 10.5 months, assessments of time to diseaseprogression (TTP in months) and response rates (RR) showed a significantaugmentation of the chemotherapeutic effect by HERCEPTIN®, withoutincrease in overall severe adverse events (AE):

Enrolled TTP (months) RR (%) AE (%) CRx 234 5.5 36.2 66 CRx + H 235 8.6*62.00** 69 AC 145 6.5 42.1 71 AC + H 146 9.0 64.9 68 T 89 4.2 25.0 59T + H 89 7.1 57.3 70 *p < 0.001 by log-rank test **p < 0.01 by X² testCRx: chemotherapy AC: anthracycline/cyclophosphamide treatment H:HERCEPTIN® T: TAXOL®

A syndrome of myocardial dysfunction similar to that observed withanthracyclines was reported more commonly with a combined treatment ofAC+H (18% Grade 3/4) than with AC alone (3%), T (0%), or T+H (2%).

These data indicate that the combination of anti-ErbB2 antibodytreatment with chemotherapy markedly increases the clinical benefit, asassessed by response rates and the evaluation of disease progression.However, due to the increased cardiac side-effects of doxorubicin orepirubicin, the combined use of anthracyclines with anti-ErbB2 antibodytherapy is contraindicated. The results, taking into account risk andbenefit, favor the combined treatment with HERCEPTIN® and paclitaxel(TAXOL®).

The disclosures of all citations in the specification are expresslyincorporated herein by reference.

1-15. (canceled)
 16. A method for the treatment of breast cancer,comprising administering to a human patient with breast cancercharacterized by overexpression of ErbB2 receptor, an effective amountof a combination of a humanized anti-ErbB2 antibody that binds toepitope 4D5 within the ErbB2 extracellular domain sequence, asdetermined by a cross-blocking assay using said antibody and antibody4D5 obtainable from deposit ATCC CRL 10463, and a taxoid, in the absenceof an anthracycline derivative, under warning instructions providingthat the combination is not to be used with an anthracycline-typechemotherapeutic agent.
 17. The method of claim 16, wherein said breastcancer is metastatic breast cancer.
 18. The method of claim 17, whereinsaid breast cancer is metastatic breast carcinoma.
 19. The method ofclaim 17, wherein the metastatic breast cancer is characterized by 2+ to3+ overexpression of the ErbB2 receptor, as determined byimmunohistochemistry or fluorescence in situ hybridization (FISH). 20.The method of claim 17, wherein the metastatic site is characterized by2+ to 3+ overexpression of the ErbB2 receptor.
 21. The method of any oneof claims 16 to 20, wherein the humanized anti-ErbB2 antibody is rhuMAbHER2.
 22. The method of any one of claims 16 to 20, wherein the taxoidis paclitaxel.
 23. The method of claim 21, wherein the taxoid ispaclitaxel.
 24. The method of claim 17, wherein said human patient didnot receive prior cytotoxic chemotherapy for treatment of metastaticbreast cancer.
 25. The method of claim 24, wherein said human patientreceived prior cytotoxic chemotherapy in the adjuvant setting.
 26. Themethod of claim 25, wherein said human patient received prior hormonaltherapy for treatment of metastatic breast cancer.
 27. The method ofclaim 26, wherein said hormonal therapy comprises administration oftamoxifen.
 28. The method of claim 16, wherein the combinedadministration comprises coadministration using separate formulations ora single formulation or the anti-ErbB2 antibody and the taxoid.
 29. Themethod of claim 16, wherein the combined administration comprisesconsecutive administration or the anti-ErbB2 antibody and the taxoid, ineither order.
 30. The method of claim 16, wherein the combinedadministration comprises simultaneous administration of the anti-ErbB2antibody and the taxoid.